Micromechanical measurements in living embryos

胚胎是一个复杂的系统，其中局部组织位移和变形是通过复合组织的大部分未知的机械特性耦合的局部和远距离力产生机制的结果。一个特别的例子是神经形成，通过这个过程，称为神经外胚层的早期细胞片层折叠成三维结构，这是脊椎动物中枢神经系统生长的框架。在最简单的情况下，比如脊髓中的神经形成，这个过程包括将一张纸大致折叠成一个圆柱体，但即使是这个过程也知之甚少。大脑中的神经形成要复杂得多，但对我们来说是必不可少的;其形态发生的错误是导致衰弱和致命的出生缺陷的根本原因。由于新的成像和图像处理技术，我们在开发捕捉细胞和组织运动的方法方面取得了长足的进步。使用全细胞跟踪和计算分析分析的三维时间推移图像显示了丰富的组织重塑谱。然而，尽管这显然是复杂的计划，我们认为，这些模式可能源于一个精心策划的一系列刻板的力量产生机制，在空间和重叠的影响模式。我们已经可以预测这些可能如何起作用，但要验证这些模型并进一步取得进展，我们需要更深入地了解组织在发育过程中不断变化的物理特性。生物学家需要在动物胚胎内存在的复杂和不断变化的条件下解决这些问题的工具。我们的目标是开发这样一种工具，并使用它来研究活跃的过程之间的平衡和潜在的机械性能的发育组织，这是必不可少的塑造正确的形态胚胎。我们计划在这个项目中开发一种微创工具，能够探测局部机械响应的活组织。该设备将是相对便携的，可安装在标准显微镜载物台上。它将在位于生物样品中的铁磁珠上施加受控的力。要控制的力的方向和大小。我们的初步测试表明，这样的实验在斑马鱼胚胎中是可以实现的。动物正常发育，这些颗粒在适当的位置和适度的磁场可以用来轻轻取代这些胚胎内的珠。这种方法将使我们能够研究发育生物学中尚未探索的领域。首先，我们将研究正常发育的胚胎中活组织的弹性和粘性/塑性特性。这一信息对于确定观察过程所需的力的范围和区分可能的机制是重要的。我们将把注意力集中在发育过程中组织成熟的分析上。从囊胚到原肠胚的转变是一个很好的例子，细胞被认为逐渐形成更紧密的连接。我们将跟踪组织力学特性随发育时间的演变，并询问这种时间变化是否是正常发育的关键。利用对许多胚胎的统计，我们将能够研究机械性能的空间模式。我们将更具体地说明大脑发育中涉及的模式有多少是由于被动特性的变化，以及主动过程和周围组织之间的平衡对正常发育至关重要。这些问题和方法，在这里开发，以解决他们适用于大多数形态发生的转变，并预计将在其他地方高度相关。

The embryo is a complex system wherein local tissue displacement and deformation is the result of local and distant force-generating mechanisms coupled through the largely-unknown mechanical properties of the composite tissues. One particular case in point is that of neurulation, the process by which the early sheet of cells, called the neural ectoderm, folds itself into the three dimensional structure that is the framework upon which the vertebrate central nervous system grows. At its simplest, such as neurulation in the spinal cord, the process involves the folding of a sheet roughly into a cylinder but even that is poorly understood. Neurulation in the brain is far more complex but essential for us to understand; errors in its morphogenesis are the root cause of debilitating and fatal birth defects. Thanks to novel imaging and image processing technologies, we have made great strides in developing methods to capture the movements of cells and tissues. Three-dimensional time-lapse images, analysed using in toto cell tracking and computational analyses show a rich spectrum of tissue remodelling. However, despite this apparently complex scheme, we believe that these patterns could originate from a well-orchestrated series of stereotypical force-generating mechanisms that are patterned in space and overlapping in their influence. We can already make predictions of how these may act but to verify these models and progress further we need far greater insight into the changing physical properties of tissues as they develop. Biologists are in need of tools to address such problems in the context of the complex and changing conditions that exist within the animal embryo. Our aim is to develop such a tool and use it to study the balance between active processes and the underlying mechanical properties of developing tissues that is essential in shaping correct morphogenesis of the embryo.We plan in this project to develop a minimally-invasive tool able to probe the local mechanical response of living tissues. The device will be relatively portable, mountable on a standard microscope stage. It will impose a controlled force upon a ferromagnetic bead located in the biological sample. The direction and magnitude of force to be controlled. Our preliminary tests have demonstrated that such an experiment is achievable in zebrafish embryos. Animals develop normally with these particles in place and modest magnetic fields can be used to gently displace beads within these embryos. This methodology will enable us to investigate largely unexplored areas of developmental biology. First, we will characterise for the the elastic and viscous/plastic properties of living tissues within a normally developing embryo. This information is important to establish the range of forces required to observed processes, and to discriminates between possible mechanisms. We will focus our attention to the analysis of tissue maturation during development. The transition from blastula to gastrula is a good example where cells thought to progressively form tighter junctions. We will follow the evolution of the tissue mechanical properties with developmental time and ask if this temporal variation is key to normal development. Using statistics on many embryos, we will be able to study spatial patterns of mechanical properties. We will more specifically characterise how much of the patterning involved in brain development is due to variations in passive properties, and how the balance between active processes and the surrounding tissue is critical for normal development. Such questions and the methodology developed here to address them apply to most morphogenetic transformations in and are expected to be highly relevant elsewhere.